B.V. Sunil Kumar

Heterologous Expression and Functional Characterization of Matrix Metalloproteinase-11 From Canine Mammary Tumor

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzymes caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.

Cyclical changes in collagen concentration in relation to growth and development of buffalo corpus luteum

In the present study, changes in luteal fresh weight and concentration of collagen in cyclic buffalo corpus luteum were investigated at 4 stages of its growth and development/regression. The collagen concentration was determined by estimating hydroxyproline, a collagen specific amino acid present in luteal tissues. The mean fresh weight increased (P < 0.001) over the luteal phase, reached maximum at late-luteal stage and decreased (P < 0.001) subsequently at follicular stage. The weight of the mature CL was 2.5 times heavier than the CL haemorrhagicum and regressing CL. Results showed that cyclic buffalo CL contains collagen at all 4 stages of development during oestrous cycle. The collagen in luteal tissues constitutes about 0.9% to 1.2% of luteal fresh weight with the highest content appearing in mature tissue. The concentration of collagen increased (P < 0.001) with the stages of CL development over the luteal phase and the highest concentration was recorded at follicular phase with the decline of luteal weight. The total content of collagen per CL also showed the same trend during luteal phase but decreased at follicular phase with the loss of luteal tissues. In conclusion, collagen concentration in cyclic buffalo CL changes with the growth and development of CL across the oestrous cycle. The synthesis of collagen is faster between early- to mid-luteal stage than mid- to late-luteal stage.

Identification of single nucleotide polymorphism in stromelysin-3 catalytic domain in canine mammary tumours

Matrix metalloproteinases (MMPs) belong to a large family of calcium-dependent, zinc-containing endopeptidases, which are responsible for tissue remodelling and degradation of extracellular matrix, including collagens, elastins, gelatin, matrix glycoproteins and proteoglycan. Certain MMPs such as stromelysin-3 (MMP11) and matrilysin (MMP7) are reported to be highly expressed during breast cancer, so they may act as marker enzymes for breast cancer progression. The genetic variation in the gene encoding catalytic domain of stromelysin-3 was investigated in healthy dogs (n=20) and dogs with mammary tumours (n=20), using an optimised non-radioactive polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) analysis. Two different SSCP patterns were detected from the 313 bp fragment. Sequencing of the gene fragment revealed a point substitution mutation in catalytic domain of stromelysin-3 in tumour subjects.

Molecular cloning and characterisation of the tissue inhibitor of metalloproteinase-3 gene from canine mammary tumour

Inhibition of matrix metalloproteinases (MMPs) by tissue inhibitors of metalloproteinases (TIMPs) has been reported to decrease metastasis and tumour-associated angiogenesis. The present study is aimed at characterising the gene encoding TIMP-3 from canine mammary tumour. We identified and cloned the full-length cDNA encoding TIMP-3 from canine mammary tumour. The entire open reading frame consisted of 636 nucleotides and 213 residues (accession number JF508171). Nucleotide and translated protein sequence were close to Sus scrofa but clustered away from the rodent group. Synonymous substitution (dS) was higher than non-synonymous substitution (dN), suggesting that the TIMP-3 gene was not under positive selection and that no advantageous mutations had any significant role in its evolutionary adaptation. The predicted SWISS-MODEL of canine TIMP-3 protein is composed of 7 α-helices and 12 β-turns. The G-factor score for the TIMP-3 protein model based on Ramachandran plot was found to be −0.47 for dihedral bonds, −0.08 for covalent bonds and −0.31 overall, which suggests that the model obtained for TIMP-3 is a reliable one.